Abstract

The importance of individual heterogeneity within a genetically identical population has become well recognized. However, single bacteria studies have been beset by a number of challenges ranging from single-cell handling to detection. Most of these stem from bacteria’s microscale dimensions and the complexity of their natural environment. In recent years, microfluidics has emerged as a powerful tool to manipulate single cells and their immediate microenvironments and is well suited to address these challenges. The protocols below will describe the creation of microfluidic devices for monolayer cell culture and long-term tracking of morphological dynamics from individual bacteria under precisely delivered perturbations. Step-by-step procedures for on-chip assays and morphological-based image analysis are described in detail, and these approaches enable fast quantification of bacteria growth and morphological changes under a broad range of conditions within a single experiment. Importantly, these methods do not require labeling of cells, thereby offering unique advantages in the investigation of naturally occurring microbes.